ACE Inhibitory Activity and Molecular Docking of Gac Seed Protein Hydrolysate Purified by HILIC and RP-HPLC

Abstract

Gac (Momordica cochinchinensis Spreng.) seed proteins (GSPs) hydrolysate was investigated for angiotensin I-converting enzyme (ACE) inhibitory activities. GSPs were hydrolyzed under simulated gastrointestinal digestion using a combination of enzymes, including pepsin, trypsin, and chymotrypsin. The screening of ACE inhibitory peptides from GSPs hydrolysate was performed using two sequential bioassay-guided fractionations, namely hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). Then, the peptides in the fraction with the highest ACE inhibitory activity were identified by LC-MS/MS. The flow-through (FT) fraction showed the most potent ACE inhibitory activity when HILIC fractionation was performed. This fraction was further separated using RP-HPLC, and the result indicated that fraction 8 (RP-F8) showed the highest ACE inhibitory activity. In the HILIC-FT/RP-F8 fraction, 14 peptides were identified using LC-MS/MS analysis coupled with de novo sequencing. These amino acid chains had not been recorded previously and their ACE inhibitory activities were analyzed in silico using the BIOPEP database. One fragment with the amino acid sequence of ALVY showed a significant ACE inhibitory activity (7.03 ± 0.09 µM). The Lineweaver-Burk plot indicated that ALVY is a competitive inhibitor. The inhibition mechanism of ALVY against ACE was further rationalized through the molecular docking simulation, which revealed that the ACE inhibitory activities of ALVY is due to interaction with the S1 (Ala354, Tyr523) and the S2 (His353, His513) pockets of ACE. Bibliographic survey allowed the identification of similarities between peptides reported as in gac fruit and other proteins. These results suggest that gac seed proteins hydrolysate can be used as a potential nutraceutical with inhibitory activity against ACE.